GSM7710862: CD4_31_14; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMC) were obtained by HIsopaque-1077-based density centrifugation (Sigma-Aldrich, St. Louis, MO,USA). CD4 and CD8 T-cell isolation from PBMC was performed via microbead-based separation (CD4 MicroBeads, human, # 130-045-10; CD8 MicroBeads, human # 130-045-201; Miltenyi Biotec, Bergisch Gladbach,Germany) via an AutoMACSPro Separator according to the manufacturer's protocol (# 130-092-545, Miltenyi Biotec, Bergisch Gladbach, Germany).T cells were lysed in QIAzol Lysis Reagent and isolated using the miRNeasy Mini Kit (50) according to the manufacturer's protocol. Messenger RNAwas purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNAwas synthesized using random hexamer primers,followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection,USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.